DNA structure has recently emerged as one of the key factors governing the ability of 5-bromodeoxyuridine (BrdU) to radiosensitize DNA. Here, we report the dependence of the specific damage induced by BrdU for different DNA conformations. Strand breaks are specific for B-form DNA, whereas A-DNA only undergoes formation of piperidine-sensitive DNA lesions. Interstrand cross-links are only found in semi-complementary B-DNA. DNA conformation was altered by gradually rehydrating lyophilized DNA samples, which induces an A- to B-form transition. These results were also validated by irradiating DNA in solution, in the presence or absence of 80% ethanol to induce an A- or B-form, respectively. Alkali-labile DNA lesions were revealed using hot piperidine to transform both base and sugar lesions into strand breaks. We also analyzed the location of damage as a function of DNA structure: piperidine-sensitive lesions were observed exclusively at the site of BrdU substitution, whereas strand breaks were able to migrate along the DNA strand, with a clear preference for the adenine 5' of the BrdU. Thus, not only the hybridization state but also the DNA conformation affect the degree of sensitization by BrdU by influencing the amount and type of damage produced. Although clinical trials using BrdU as a radiosensitizer have been disappointing up to this point, these new findings point to several key features of BrdU radiosensitization that may alter future radiotherapeutic studies.
Interstrand cross-links impede critical cellular processes such as transcription and replication and are thus considered to be one of the most toxic types of DNA damage. Although several studies now point to the existence of gamma-radiation-induced cross-links in cellular DNA, little is known about the characteristics required for their creation. Recently, we reported the formation of interstrand cross-links that were specific for mismatched nucleotides within 5-bromo-2'-deoxyuridine-substituted DNA. Given the structural specificity for interstrand cross-link formation, it is likely that open or mismatched regions of DNA in cells may be particularly favorable for cross-link production. Herein, we investigated the effect of the local DNA sequence on the formation of interstrand cross-links, using 5-bromo-2'-deoxyuridine to generate radicals in a mismatched region of DNA. We investigated a total of 12 variations of bases in the mismatched region. The oligonucleotides were irradiated with gamma-rays, and interstrand cross-link formation was analyzed by denaturing gel electrophoresis. We found that the efficiency of cross-link formation was highly dependent on the nature of mismatched bases and, on the basis of electrophoretic mobility, observed several distinctive cross-link structures with specific DNA sequences. This study provides new insights into the reactivity of mismatched DNA and the mechanisms leading to interstrand cross-link formation. The potential application of 5-bromo-2'-deoxyuridine-induced interstrand cross-links to the field of DNA repair is discussed.
Self-assembled monolayers of 5'-32P-labeled 3'-thiolated oligonucleotides chemisorbed on gold were bombarded by low-energy electrons (LEE) of 8-68 eV. Shorter 5'-32P-oligonucleotides produced by LEE-induced strand breaks were separated with denaturing polyacrylamide gel electrophoresis and quantified by phosphor imaging. The yields of short oligonucleotides (y) decrease exponentially with their length (n), following the equation y=ae-bn, where a and b are constants, which are related to the average effective cross section per nucleotide for DNA strand break (sigmaeff) and the attenuation length (AL=1b) of LEE, respectively. The AL decreases with LEE energies from 2.5+/-0.6 nm at 8 eV to 0.8+/-0.1 nm at 68 eV, whereas sigmaeff increases from (3+/-1)x10(-18) to (5.1+/-1.6)x10(-17) cm2 within the same energy range. The energy dependence of sigmaeff shows a resonance peak of (2.8+/-0.9)x10(-17) cm2 at 18 eV superimposed on a monotonically rising curve. Transient electron attachment to a sigma* anion state of the deoxyribose group, followed by dipolar dissociation into H- and the corresponding positive-ion radical, leading to C-O bond cleavage, is proposed to account for this maximum.
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